Pluripotent human embryonic stem cells and induced pluripotent stem cells provide limitless cell sources for tissue regeneration (Thomson et al., 1998. Science 282:1145-7; Okita al., 2007, Nature 448:313-7; Takahashi et al., 2006, Cell 126:663-76; Yu et al, 2007, Science 318:1917-20). However, both confer a risk of forming tumors, which must be addressed before their clinical applications are feasible. While in most cases embryonic stem cells and induced pluripotent stem cells will be differentiated into target cells before they are transplanted to patients, the risk of tumor formation is still significant, since residual pluripotent stem cells will remain even after selection based on cell surface markers. Several groups observed teratoma formation after transplantation of differentiated cells from pluripotent stem cells, due to the presence of a low percentage of undifferentiated pluripotent stem cells (Hentze et al., 2009, Stem Cell Res 2:198-210; Xu et al., 2008, Cytotherapy 10:376-89; Duinsbergen et al., 2009, Ann N Y Acad Sci 1176:197-204). Even though 100% of the stem cells are differentiated, in vivo dedifferentiation into pluripotent stem cells is still possible (Brawley et al., 2004, Science 304:1331-4). Thus, eliminating residual pluripotent stem cells from differentiated cells in vitro and in vivo will reduce the risk of tumor formation and is highly desirable (Kiuru et al., 2009, Cell Stem Cell 4:289-300).
Genetic modification of pluripotent stem cells by suicide genes has been explored to reduce the risk of tumorigenicity. Teratomas formed by embryonic stem cells constitutively expressing the herpes simplex virus thymidine kinase (tk) gene were inhibited by ganciclovir delivery (Schuldiner et al., 2003, Stem Cells 21:257-65; Cao et al., 2006, Circulation 113:1005-14; Jung et al., 2007, Hum Gene Ther 18:1182-92). The pluripotent stem cell-specific OCT4 or NANOG promoter was subsequently used to selectively remove undifferentiated cells from differentiated cells (Naujok et al., 2010, Stem Cell Rev 6:450-61; Hara et al., 2008, Stem Cells Dev 17:619-27; Cheng et al., 2012, Biomaterials 33:3195-204). Other toxic proteins, such as α-1,3-galactosyltransferase and inducible caspase-1, have also been tested to eliminate malignant cells (Hewitt et al., 2007, Stem Cells 25(1):10-8; Wang et al., 2012, Stern Cells 30:169-79). In all these studies, transgenes were integrated into the genome by plasmid or lentiviral integration. This again raises safety concerns due to the possibility of inactivation of tumor suppressor genes or activation of oncogenes (Nusse et al., 1984, Nature 307:131-6). In addition, integrated genes are subject to position effects and silencing (Ellis, 2005, Hum Gene Ther 16:1241-6), making their expression unreliable and unpredictable. Efforts are being made to identify and validate genomic safe harbors for transgene integration to minimize the risks described above (Sadelain et al., 2012, Nat Rev Cancer 12:51-8).
Plasmid vectors containing the scaffold/matrix attached regions (S/MAR) of the human interferon-β gene can maintain their state as episomal DNA in cells of various species, and the plasmid DNA replicates during cell division if the S/MAR sequence is transcribed (piechaczek et al., 1999, Nucleic Acids Res 27:426-8; Stehle et al., 2003, Chromosome Res 11:413-21; Manzini et al., 2006, Proc Natl Acad Sci USA 103:17672-7; Jenke et al., 2004, Proc Natl Acad Sci USA 101:11322-7). The vector replicates once per cell cycle during early S-phase, with the origin recognition complex assembled at various regions on the vector DNA (Schaarschmidt et al., 2004, EMBO J. 23:191-201). The origin recognition complex stably interacts with metaphase chromosomes, which leads to stable episomal maintenance (Baiker et al., 2000, Nat Cell Biol 2:182-4):
Since S/MAR-based vectors do not integrate into the genome of mammalian cells and mediate long-term acne transfer, they should carry reduced risks of insertional mutagenesis, silencing and variegation. However, in currently available S/MAR-based vectors, the S/MAR sequence is transcribed by the same promoter driving the expression of target genes, so that they can only maintain an episomal state in cells expressing the target gene.
Thus, there is a need in the art for an S/MAR based construct to maintain its long term episomal state in all cells, while expressing a target gene only in a subset of cells. The present invention satisfies this need.